A20, a novel Zn-finger protein, was first described as a TNF-inducible early response gene cloned from human umbilical vein endothelial cells (HUVEC). Its only previously described function was its ability to inhibit TNF-induced apoptosis in a variety of human cell types. This effect has not yet been demonstrated in the endothelial cell (EC). In recent work, we have described a new function for A20 i.e., inhibition of EC activation. We demonstrated that A20 expression inhibits gene upregulation induced by a broad spectrum of EC agonists: TNF, LPS, PMA and H202 shown by transfecting bovine aortic EC with A20 and reporter constructs consisting of promoters of genes known to be upregulated during EC activation, i.e., E-selectin, IL-8 and IkBa. The inhibitory action of A20 is in part, or totally, due to blockade of NF-kB. This effect is specific, as A20 does not block the non-inducible, constitutively expressed reporter, RSVLuc nor does it block the NF-kB independent induction of the HIV-CAT reporter by the viral protein c-Tat. Interestingly, other anti-apoptotic genes belonging to the bcl gene family, namely bcl-2, bcl-xl and A1 have the same effect as A20; i.e., inhibition of EC activation through inhibition of NF-kB. Using adenovirus-mediated gene transfer, we were able to show that A20, as well as all the anti-apoptotic bcl genes, inhibit NF-kB activation at a level upstream of IkBa phopsphorylation and degradation. Taken together, these results suggest that in EC anti-apoptotic genes cross talk with the transcriptional machinery by blocking Nf-kB and thus function as down-modulators of EC activation. In addition, while blockade of NF-kB by overexpression of IkB( sensitizes EC to TNF-mediated apoptosis, we show for the first time that A20 retains its anti-apoptotic function and not only protects cycloheximide-sensitized EC from TNF-mediated apoptosis but also rescues IkBa-expressing EC from death. This result together with the expression of A20 in EC of long term surviving hamster to rat heart xenograft, qualify the therapeutic potential of A20. The aim of this study is to dissect the structure-function relationship of the inhibitory effect of A20 upon EC activation and to evaluate its anti-apoptotic role in EC. During these five years, we hope to gain insights into its mechanism of action and the means by which A20 inhibits NF-kB and interferes with signaling pathways in EC. We will try to identify the functional domains of A20 that sustain its inhibitory and its anti-apoptotic effects and whether these domains implicate interaction with specific proteins involved in signaling pathways.